Trafficking between the nucleus and cytoplasm occurs through the nuclear pore complexes (NPCs). During mitosis, metazoan NPCs disassemble into approximately a dozen subunits. Two of these subunits are targeted to mitotic kinetochores: First, the RanBP2 complex associates with kinetochores in a microtubule-dependent manner. This complex consists of RanBP2 (a large nucleoporin that is also known as Nup358), SUMO-1-conjugated RanGAP1 (the activating protein for the Ran GTPase) and Ubc9 (the sole conjugating enzyme for the SUMO family of ubiquitin-like modifiers). Second, the nine protein vertebrate Nup107-160 complex associates to kinetochores throughout mitosis in a microtubule-independent manner. The Nup107-160 complex includes Nup160, Nup133, Nup107, Nup96, Nup85, Nup43, Nup37, Sec13, and Seh1. During telophase, Nup107-160 is targeted to chromosomes and it acts in a critical and early fashion during NPC re-assembly.[unreadable] The nucleoporin RanBP2 resides on the cytoplasmic face of the interphase nuclear pore complex (NPC). Notably, a fraction of endogenous RanBP2 interacts with interphase microtubules through its N-terminal region (BPN). Overexpression of the BPN domain of RanBP2 causes dramatic increases in microtubule bundling and stability. Ectopic expression of BPN and full-length RanBP2 caused increased levels of acetylated and detyrosinated alpha-tubulin, two markers for stable microtubules. Under the same circumstances, microtubules showed resistance to nocodazole, a depolymerizing agent. Depletion of RanBP2 disrupted polarized stabilization of microtubules during directed cell migration, showing RanBP2 in regulates interphase microtubules under physiological conditions. During metaphase, RanBP2 complex recruitment to kinetochores is important for correct microtubule-kinetochore attachment. Our earlier studies suggested that complex targeting occurs through the RanBP2 protein, and that its recruitment is both regulated by and essential for correct microtubule-kinetochore attachment. We further found that the Crm1 nuclear export receptor localizes to kinetochores and promotes the binding of the RanBP2 complex. We are currently examining novel RanBP2-binding components and their role in regulation of RanBP2s activity in microtubule dynamics during both interphase and mitosis.[unreadable] Nup107-160 is broadly distributed on spindles during prometaphase. It remains kinetochore-bound throughout mitosis, and shows enhanced accumulation on unattached kinetochores. Our earlier findings showed that Nup107-160 promotes spindle assembly in a context that is distinct from intact interphase NPCs. We have recently found that Nup107-160 interacts with an active form of the gamma-tubulin ring complex (gamma-TuRC), an essential and conserved microtubule nucleator. Like Nup107-160, gamma-TuRC localized to unattached mitotic kinetochores, prompting us to test whether Nup107-160 and gamma-TuRC might function in a coordinated manner to promote nucleation of microtubules near mitotic chromosomes and at kinetochores. Xenopus egg extracts lacking the Nup107-160 complex or gamma-TuRC failed to assemble spindles around sperm chromatin or DNA beads. Moreover, HeLa cells lacking Nup107-160 or gamma-TuRC were profoundly deficient in kinetochore-associated microtubule nucleation. Our findings indicate that Nup107-160 promotes spindle assembly through the regulated nucleation of microtubules by gamma-TuRC at kinetochores and perhaps other sites within mitotic spindles. These observations suggest an important and novel relationship between the NPC and the microtubule cytoskeleton.[unreadable] Our future studies will focus on a number of issues, including the component(s) at kinetochores that is directly involved in Crm1 recruitment, the relationship between the Nup107-160 and RanBP2 complexes during mitosis, and how they together regulate the attachment of microtubules to kinteochores and the spindle assembly checkpoint. We are also examining the mitotic localization and function of other NPC components.